Project 2 Aim 1. To investigate biologic consequences of TrkB/PI-3Kinase/AKT activation on NB tumor biology Inhibition of AKT pathway in neuroblastoma inhibits tumor cell growth in vitro and in vivo. Our studies have shown that it is reasonable to pursue inhibition of the TrkB receptor as an adjunct to therapy and that the use of an Akt inhibitor would be valuable in combination or following receptor targeted therapy. 1. TrkB activation contributes to chemo-resistance in neuroblastoma (NB). We evaluated the effect of a Trk tyrosine kinase inhibitor, AZD6918 on the sensitivity of TrkB-expressing NB cells or tumors alone and in combination with etoposide, a topoisomerase II inhibitor. AZD6918 induced cell death as a single agent and attenuated BDNF/TrkB-induced protection from etoposide in vitro. Although AZD6918 alone didn't show anti-tumor growth effect or survival advantage in vivo, a combination of AZD6918 and etoposide had a statistically significant stronger anti-tumor growth effect and survival advantage compared to treatment with either agent alone. These data extend the spectrum of cytotoxic drugs whose efficacy is increased in combination with Trk inhibitors and support the combination of Trk inhibitors and cytotoxics for NB treatment. These data were recently published in Li et al Cancer Biol Ther. 16:477-83, 2015. 2. Our previous studies have identified activation of the PI3Kinase/Akt/GSK3B pathway mediates resistance to chemotherapy in neuroblastoma cells. Moreover our genetic and pharmacologic studies indicated that activation of AKT alone attenuated the effects of chemotherapy in neuroblastoma cells. Since activated AKT is more highly expressed in tumors of Neuroblastoma patients with a poor prognosis, we screened a number of inhibitors of the AKT that could enhance the efficacy of chemotherapy in our pre-clinical in vitro models. since targeting receptors may cause activation of AKT due to feedback suppression. Our studies on the AKT allosteric inhibitor MK2206, showed that inhibition of AKT activity as assessed by inhibition of phosphorylation of downstream target S6 kinase in the pharmacologic range but the growth of only 3/9 NB cell lines tested is inhibited within this range. We therefore investigated whether MK-2206 increased the sensitivity of NB to etoposide or rapamycin. Here we showed that in vitro, a synergistic effect was detected in combination of MK-2206 with etoposide through apoptosis, and MK-2206 enhanced the sensitivity to rapamycin via induction of reactive oxygen species; in vivo, a significant increased anti-tumor growth effect and murine survival advantage were observed in the combination of MK-2206 with etoposide or rapamycin. Of interest was our finding that MK-2206 and rapamycin generated ROS in MYCN amplified NB tumors and not MYCN-wt tumors, suggesting this combination therapy may be more efficacious for MYCN-ampilfied tumors. Specific Aim 2. To investigate STAT3 activation and NB tumor biology. In a recently published study, we evaluated the involvement of aberrantly activated IL-6/JAK/STAT pathway in Neuroblastoma, Rhabdomyosarcoma and Ewings Sarcoma. We identified that this pathway was activated in these pediatric tumors and could be inhibited using a JAK1/2 selective inhibitor AZD1480. Our data indicate that AZD1480 blocks the constitutive endogenous as well as cytokine-induced STAT3 activation. AZD1480 treatment decreased cell viability in 7/7NB, 7/7RMS and 2/2 EWS cell lines tested (median EC50 is 1.5uM, ranging from 0.36-5.37uM). Demonstration of in vitro and in vivo anti-tumor activity of AZD1480 provides and extends the rationale for the clinical evaluation of AZD1480 in the treatment of pediatric patients with solid malignancies. A limitation of this study is the off-target effects of AZD1480 and toxicity in the Phase I study led to discontinuation of the development of this drug. 2. Evaluation of genetic inhibition of STAT3. We have continued pre-clinical studies evaluating genetic inhibition of STAT3 using tet-regulated shSTAT3 expression vectors. These studies are complemented by ones in which we will evaluate a first in class siRNA targeting STAT3. However, there are significant off-target effects of AZD1480 and so to specifically evaluate STAT3 expression and activity in neuroblastoma we are currently evaluating AZD9150, a first in class anti-sense oligomer targeting human STAT3 for its effects on the growth of neuroblastoma tumor cells in our pre-clinical models.